Merkel Cell Carcinoma (MCC) is an aggressive virus-associated skin cancer with a disease- specific mortality of 46% and few effective treatments for advanced disease. Over 80% of MCCs are caused by persistent expression of truncated T-antigen oncoproteins from the Merkel polyomavirus (MCPyV). The immune system is particularly important in MCC, as there is a higher incidence of MCC in immunosuppressed people and >50% of patients mount lymphocyte responses to the T- antigen. Similar to other cancers, infiltration of CD8 T cells into the tumor is associated with improved MCC-specific survival. The well-established involvement of the adaptive immune response to non- self tumor antigens in MCC has led to clinical trials of many immune-stimulating therapies for these patients. Our extensive and unique repository of MCC blood and tumor specimens, including samples from patients receiving any of seven immune-targeted treatments undergoing clinical investigation with variable efficacy, will allow us to further characterize the immune response to MCPyV and to investigate mechanisms of therapeutic action. We hypothesize that both T cell-targeted and general immunotherapies will cause phenotypic and functional changes in MCPyV-specific T cells (addressed in Aim 1). Flow cytometry will be used to measure both inhibitory surface markers and effector cytokine secretion by MCPyV-specific tetramer-positive cells. By measuring effector cytokine secretion of patient blood and tumor- infiltrating lymphocytes after stimulation with pools of MCPyV T-antigen peptides, we will investigate whether broadening of the T lymphocyte response to additional MCPyV epitopes occurs post-therapy (Aim 2). In addition, we propose to characterize the T cell receptors (TCRs) recognizing one common HLA type/MCPyV epitope by limiting dilution cloning in conjunction with high-throughput sequencing of tetramer-positive T cells from several MCC patients. We will determine the genetic and functional diversity of the TCR repertoire recognizing this epitope as we search for public responses to MCPyV and for a highly avid TCR (Aim 3). These studies should help us define the mechanisms by which some patients have superior disease outcomes, and contribute to our goal of improving therapeutic options for patients with advanced MCC.